Stevens J L, Ayoubi N, Robbins J D
W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946.
J Biol Chem. 1988 Mar 5;263(7):3395-401.
The submitochondrial localization and identity of enzymes which metabolize cysteine conjugates were investigated. Glutamine transaminase K was purified from rat kidney mitochondrial soluble fraction and was shown to be a cysteine conjugate beta-lyase. The purified mitochondrial enzyme is similar to the cytosolic glutamine transaminase K whose beta-lyase activity with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is regulated by concurrent transamination (Stevens, J. L., Robbins, J. D., and Byrd, R. A. (1986) J. Biol. Chem. 261, 15529-15537). However, beta-lyase activity in whole mitochondria is largely independent of regulation by cosubstrates for transamination suggesting that factors present in mitochondria are able to support the beta-lyase activity in the absence of added alpha-keto acid. Fractionation of mitochondria results in a loss of the independent beta-lyase activity. However, the majority of the beta-lyase activity can be recovered in the matrix if it is stimulated by the addition of alpha-keto-gamma-methiolbutyrate. The data suggest that the regulation of beta-elimination by the competing transamination pathway is different for each substrate and that multiple beta-lyases may exist in rat kidney. S-(2-Benzothiazolyl)-L-cysteine (BTC) is a poor substrate for purified glutamine transaminase K from mitochondria and cytosol, but BTC is as active as DCVC in crude mitochondrial matrix suggesting that other enzymes may be present. In contrast to DCVC, with BTC as substrate, the beta-lyase activity of the purified enzyme and enzyme(s) in the mitochondrial matrix is largely alpha-keto acid-independent. The existence of multiple enzymes is also supported by the observation that alpha-keto acids which are not substrates for purified glutamine transaminase K from mitochondria and cytosol do stimulate beta-lyase activity in the mitochondrial matrix fraction. Mitoplasts were found to be sensitive to DCVC toxicity consistent with the matrix localization of beta-lyase activity. The possible role in cysteine conjugate toxicity of matrix enzyme regulation by alpha-keto acids is discussed.
对代谢半胱氨酸共轭物的酶的亚线粒体定位和特性进行了研究。谷氨酰胺转氨酶K从大鼠肾脏线粒体可溶性部分中纯化出来，并被证明是一种半胱氨酸共轭β-裂解酶。纯化后的线粒体酶与胞质谷氨酰胺转氨酶K相似，其对S-(1,2-二氯乙烯基)-L-半胱氨酸（DCVC）的β-裂解酶活性受同时进行的转氨作用调节（史蒂文斯，J.L.，罗宾斯，J.D.，和伯德，R.A.（1986年）《生物化学杂志》261，15529 - 15537）。然而，完整线粒体中的β-裂解酶活性在很大程度上不依赖于转氨作用共底物的调节，这表明线粒体中存在的因子能够在不添加α-酮酸的情况下支持β-裂解酶活性。线粒体分级分离导致独立的β-裂解酶活性丧失。然而，如果通过添加α-酮基-γ-甲硫基丁酸来刺激，大部分β-裂解酶活性可以在基质中恢复。数据表明，对于每种底物，竞争性转氨途径对β-消除的调节是不同的，并且大鼠肾脏中可能存在多种β-裂解酶。S-(2-苯并噻唑基)-L-半胱氨酸（BTC）是从线粒体和胞质中纯化得到的谷氨酰胺转氨酶K的不良底物，但BTC在粗线粒体基质中的活性与DCVC相同，这表明可能存在其他酶。与DCVC相反，以BTC为底物时，纯化酶和线粒体基质中的酶的β-裂解酶活性在很大程度上不依赖于α-酮酸。多种酶的存在也得到了以下观察结果的支持：不是线粒体和胞质中纯化的谷氨酰胺转氨酶K底物的α-酮酸确实能刺激线粒体基质部分的β-裂解酶活性。发现线粒体质对DCVC毒性敏感，这与β-裂解酶活性的基质定位一致。讨论了α-酮酸对基质酶调节在半胱氨酸共轭物毒性中的可能作用。
